For many years, lactic acid bacteria have been utilized as fermenting agents for the preservation of food taking benefit of a low pH and the action of fermentation products generated during the fermentative activity thereof to inhibit the growth of spoilage bacteria. With this aim, non-well characterized lactic acid bacteria or “fermentum” have been used to prepare a variety of different foodstuffs such as dry fermented meat products, cheese, and other fermented dairy products from milk.
Recently, lactic acid bacteria have attracted a great deal of attention because some strains have been found to exhibit valuable properties to man and animals upon ingestion. In particular, specific strains of the genus Lactobacillus or Bifidobacterium have been found to be able to colonize the intestinal mucosa and to assist in the maintenance of the well-being of man and animal, and has been named such as probiotics.
Probiotics are considered to be viable microbial preparations which promote the individual's health by preserving a healthier microflora in the intestine. A microbial preparation may be commonly accepted as a probiotic in case the effectual thereof and their mode of action are known. Probiotics are deemed to attach to the intestine's mucosa, colonize the intestinal tract and likewise prevent attachment of harmful microorganisms thereon. A crucial prerequisite for their action resides in that they have to reach the gut's mucosa in a proper and viable form and do not get destroyed in the upper part of the gastrointestinal tract, especially by the influence of the low pH prevailing in the stomach. During the extensive studies leading to new probiotic strains, previous patent applications have described the isolation of variety of different bacterial strains from baby feces (JP04320642, JP05227946). Moreover, the probiotic strains obtained up to now were mainly selected for their capability to adhere to the intestinal mucosa, usually by in vitro experiments. Subsequent selection has not always been performed, and if it has, it used to be mainly based on individual properties of the strain. Finally, and sometimes after the commercialization of the strain, the beneficial effects of the selected strain have been proven in vivo.
In this regard, several patent applications such as eg. EP0768375, WO97/00078, EP0577903 and WO00/53200 disclose specific strains of Bifidibacterium and Lactobacillus and their beneficial effects on diarrhea, immunomodulation, hypersensitivity reactions or infection by pathogen microorganisms.
Moreover, the beneficial effects of human breast milk on the well-being of infants compared to those fed with milk-based formula has also been extensively reported. In this regard, a reduction in the risk of infection, allergy, asthma and related affections, and an improvement of the intestinal maturation and gut functions has been described. Also, it has been reported that the composition of the gut flora is different to human-milk fed infants from those fed with milk-based formula. The beneficial effects and the modulation of the gut flora of breast human milk have been attributed to its characteristic composition as compared to infant formulas. Thus, the benefits of breast milk proteins such as lactoferrin or maternal immunoglobulins, and the rich composition in oligosaccharides that may act as prebiotic compounds, in the regulation of the flora and gut functions have been reported.
However, to our knowledge there is no publication or work that describes the presence of microbial strains in normal human breast milk. Neither it has been reported that such microbial strains could be beneficial for the breast-fed baby, and therefore acting as probiotics modulating the gut flora of the breast feeding infant. Our work suggests that the well-being effects of the human breast-fed could be also mediated by microbial strains present therein.
It has been suggested in several works that the initial colonization of the neonate is due to cross-contamination with vaginal microflora during labor. However, there are several studies that show similar initial microbial colonization of the neonate independently of the neonate delivery route (cesarean versus natural labor). Moreover, the fact that it is not possible to obtain germ-free animals from conventional pregnant mice nevertheless they have been obtained by cesarean, and that these animals could also be obtained after embryo delivery to sterile recipient mice (Okamoto, M. and Matsumoto, T. 1999. Exp. Anim. 48: 59-62), suggest to us that it has to be other mechanisms than vaginal contamination that also influences the initial colonization of the neonate, and that this mechanism has to begin before labor.
In this regard, to our knowledge, there is no publication or work describing the presence of lactic acid bacterial strains in normal human amniotic fluid. Neither it has been reported that such non-pathogenic microbial strains could be beneficial for the gestating baby, and therefore conditioning, just during the gestation, the initial microbial populations able to colonize the fetal gut.
In understanding the valuable properties that particular strains of lactic acid bacteria may provide, there is a desire in the art for additional lactic acid bacterial strains that are beneficial to the well being of man and/or animal. Consequently, a problem of the prior art was to provide rational methods for the selection of additional new bacterial strains and novel sources for the selection of them , that allow the obtention of bacterial strains which exhibit individually a high number of beneficial properties for man and/or animals. The above problem has been solved by providing novel microorganisms, namely lactic acid bacteria, belonging to the genus Lactobacillus. These new strains have been obtained from different sources apart from feces, such as goat cheese and from human breast milk and amniotic fluid, and have been chosen by a method consisting in the ability of these strains to survive in breast milk and/or amniotic fluid, and by their ability to be transferred to breast milk and/or amniotic fluid after oral intake.
This selection method ensures that the bacterial strains obtained have implicitly most of the characteristics attributed to a potential probiotic strain, namely good resistance to digestion process and the ability of gut colonization, but also a more natural human origin, safety aspects, and the ability to colonize and regulate some human niches other than the gut. Finally, the selected strains have also been tested not only for their adhesion capabilities but for having a high degree of beneficial characteristics.